ToxSci Advance Access originally published online on April 13, 2007
Toxicological Sciences 2007 98(1):216-230; doi:10.1093/toxsci/kfm087
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Cyclophosphamide-Induced Apoptosis in COV434 Human Granulosa Cells Involves Oxidative Stress and Glutathione Depletion
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* Department of Community and Environmental Medicine
Department of Medicine, University of California, Irvine, California 92617
Department of Environmental Health, School of Public Health, Shanghai Jiaotong University, Shanghai, China
Department of Developmental and Cell Biology, University of California, Irvine, California 92617
1 To whom correspondence should be addressed at Center for Occupational and Environmental Health, 5201 California Avenue, Suite 100, Irvine, CA 92617. Fax: (949) 824-2345. E-mail: uluderer{at}uci.edu.
Received November 2, 2006; accepted April 10, 2007
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Abstract |
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The anticancer drug cyclophosphamide induces granulosa cell apoptosis and is detoxified by glutathione (GSH) conjugation. We previously showed that both cyclophosphamide treatment and GSH depletion induced granulosa cell apoptosis in rats, but the role of GSH in apoptosis in human ovarian cells has not been studied. Using the COV434 human granulosa cell line, we tested the hypotheses that (1) GSH depletion or treatment with 4-hydroperoxycyclophosphamide (4HC), a preactivated form of cyclophosphamide, induces apoptosis, (2) GSH depletion potentiates 4HC-induced apoptosis, and (3) 4HC-induced apoptosis is mediated by GSH depletion and oxidative stress. Cells were treated with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, with or without follicle stimulating hormone (FSH) or serum. A significant increase in the number of apoptotic cells, assessed by terminal deoxynucleotidyl transferase–mediated deoxy-uridine triphosphate nick-end labeling (TUNEL) and Hoechst 33342 staining, occurred with BSO treatment. Treatment with 4HC dose-dependently induced apoptosis by TUNEL, Hoechst staining, and caspase 3 activation. Treatment with 4HC caused an increase in reactive oxygen species generation, measured by dichlorofluorescein fluorescence, oxidative DNA damage, measured by 8-hydroxyguanosine immunostaining, and an oxidation of the redox potential for the oxidized glutathione/reduced glutathione couple. Total intracellular GSH declined after 4HC treatment, preceding the onset of cell death. Treatment with antioxidants inhibited 4HC-induced apoptosis. Combined treatment with BSO and 4HC caused greater induction of apoptosis than either treatment alone. These findings are consistent with roles for oxidative stress and GSH depletion in mediating the induction of apoptosis in COV434 cells by cyclophosphamide.
Key Words: granulosa cell; glutathione; cyclophosphamide; apoptosis; 4-hydroperoxycyclophosphamide; oxidative stress; ovary; antioxidant.
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